Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/07/25/jpet.116.233452.DC1 1521-0103/359/1/18–25$25.00 http://dx.doi.org/10.1124/jpet.116.233452 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 359:18–25, October 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics Propacetamol-Induced Injection Pain Is Associated with Activation of Transient Receptor Potential Vanilloid 1 Channels s Florian Schillers, Esther Eberhardt, Andreas Leffler, and Mirjam Eberhardt Department of Anaesthesiology and Intensive Care Medicine, Hannover Medical School, Hannover, Germany (F.S., A.L., M.E.); and Department of Anaesthesiology, Friedrich-Alexander University Erlangen-Nuremberg, Erlangen, Germany (E.E.) Received March 8, 2016; accepted July 22, 2016 ABSTRACT Downloaded from Propacetamol (PPCM) is a prodrug of paracetamol (PCM), were expressed in human embryonic kidney 293 cells and which was generated to increase water solubility of PCM for investigated by means of whole-cell patch clamp and ratio- intravenous delivery. PPCM is rapidly hydrolyzed by plasma metric calcium imaging. PPCM (but not PCM) activated esterases to PCM and diethylglycine and shares some struc- TRPV1, sensitized heat-induced currents, and caused an tural and metabolic properties with lidocaine. Although PPCM increase in intracellular calcium. In TRPA1-expressing cells is considered to be comparable to PCM regarding its analgesic however, both PPCM and PCM evoked calcium responses but properties, injection pain is a common side effect described for failed to induce inward currents. Intracutaneous injection of jpet.aspetjournals.org PPCM but not PCM. Injection pain is a frequent and unpleasant PPCM, but not of PCM, in human volunteers induced an intense side effect of numerous drugs in clinical use, and previous and short-lasting pain and an increase in superficial blood flow, reports have indicated that the ligand gated ion channels indicating activation of nociceptive C fibers and subsequent transient receptor potential ankyrin 1 (TRPA1) and transient neuropeptide release. In conclusion, activation of human TRPV1 receptor potential vanilloid 1 (TRPV1) can mediate this effect by PPCM seems to be a relevant mechanism for induc- on sensory neurons. This study aimed to investigate molecular tion of pain upon intracutaneous injection and thus also for mechanisms by which PPCM, in contrast to PCM, causes pain reported as an adverse side effect upon intravenous injection pain. Therefore, human TRPV1 and TRPA1 receptors administration. at ASPET Journals on September 24, 2021 Introduction intravenous application of PPCM (Depré et al., 1992). In addition to pain and local irritation at the injection site, health Paracetamol (PCM; acetaminophen) is a popular analgesic care–associated cases of allergic reactions have been reported drug for light to medium pain. However, because of its poor for PPCM (Barbaud et al., 1995). solubility and stability in water-based solutions at physiologic Injection pain is a common problem causing discomfort and pH values, it is less suitable for intravenous application. distress during induction of general anesthesia with propofol Therefore, propacetamol (PPCM), a prodrug of PCM, was (Doenicke et al., 1996; Tan and Onsiong, 1998) or etomidate generated by esterification of PCM and the carboxylic acid and even after local infiltration with local anesthetics such as diethylglycine to improve solubility (see Fig. 1 for a compar- lidocaine. Many of these proalgesic substances were described ison of molecular structures) (Barbaud et al., 1995). PPCM to activate transient receptor potential (TRP) channels was effectively used to treat fever and pain in several expressed in nociceptive nerve endings, and transient receptor European countries when oral application was not appropri- potential vanilloid 1 (TRPV1) and transient receptor potential ate. Intravenous infusion leads to rapid hydrolysis of PPCM ankyrin 1 (TRPA1) currently seem to be the most relevant within 7 minutes by plasma esterases in PCM and diethylgly- candidates mediating activation and/or sensitization of sen- cine (Bannwarth et al., 1992). Although analgesic properties of sory neurons by both propofol and local anesthetics (Leffler PPCM are proven to be superior to placebo and comparable to et al., 2008, 2011; Matta et al., 2008). Both TRPV1 and TRPA1 the effects of PCM (McNicol et al., 2011), it has become evident nonselective cation channels are expressed in peripheral that injection pain occurs quite frequently (up to 39%) upon nerve endings of nociceptive C and Ad fibers and also along the peripheral axon and in central nerve terminals in the spinal cord. As polymodal receptors, TRPA1 and TRPV1 can This research was supported by intramural grants from Hannover Medical be activated by a multitude of different agonists related to School. dx.doi.org/10.1124/jpet.116.233452. pain, inflammation, and oxidative stress. Among several s This article has supplemental material available at jpet.aspetjournals.org. mechanisms resulting in activation, the modification of ABBREVIATIONS: ANOVA, analysis of variance; BCTC, 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide; GSK1016790A, N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene- 2-carboxamide; HC030031, 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide; HEK, human embryonic kidney; HSD, honestly significant difference; hTRP, human transient receptor potential; PCM, paracetamol; PPCM, propacetamol; TRP, transient receptor potential; TRPA, transient receptor potential ankyrin; TRPV, transient receptor potential vanilloid. 18 Propacetamol Activates TRPV1 to Evoke Pain 19 (PAA, Pasching, Austria). Cells were cultured at 37°C and 5% CO2 for 24–48 hours before use for patch clamp or calcium imaging. Patch Clamp Experiments. Cells were examined by whole-cell patch clamp with an EPC10 USB HEKA amplifier (HEKA Elektronik, Lamprecht, Germany), low-passed at 1 kHz, and sampled at 2 kHz. Pipettes were pulled from borosilicate glass tubes (TW150F- 3; World Precision Instruments, Berlin, Germany) to give a resistance of 2–5MV. The external calcium-free solution included 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES, and 10 mM glucose (pH 7.4 was adjusted with tetramethylammonium hydroxide). The internal solution contained 140 mM KCl, 2 mM MgCl2,5mM EGTA, and 10 mM HEPES (pH was adjusted with KOH). Cells were either held at 260 mV or currents evoked by 500-millisecond voltage ramps from 2100 to 1100 mV were measured. All experiments were performed at room temperature. Na1 currents were recorded from the ND7/23-hybrid cell line derived from neonatal rat spinal sensory neurons fused with mouse neuroblastoma cells (Wood et al., 1990). For these experiments, data were sampled at 20 kHz and filtered at 5 kHz. The series resistance Downloaded from Fig. 1. Molecular structure of PCM and PPCM. Ester hydrolysis of the was compensated by 60%–70% and the capacitance artifacts were water-soluble prodrug PPCM yields PCM and diethylglycine. cancelled using Patchmaster software (HEKA Elektronik). Pipettes were pulled with a resistance of 1 to 2 MV and filled with internal solution containing 140 mM CsF, 2 mM MgCl2, 5 mM EGTA, and N-terminal cysteines has been shown as the underlying 10 mM HEPES (pH was adjusted to 7.4 by KOH). The external mechanism of how reactive agents sensitize and activate these solution contained 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1.2 mM ion channels (Hinman et al., 2006; Chuang and Lin, 2009). We CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4 was adjusted by jpet.aspetjournals.org noted that PPCM indeed shows some structural similarities tetramethylammonium hydroxide). Sodium currents were elicited by to lidocaine. Furthermore, PPCM is metabolized by cleavage depolarizing pulses to 210 mV at 0.1 or 10 Hz using 2120 mV as the of diethylglycine which is similar to ethylglycin generated holding potential. Fitmaster (HEKA Elektronik) and Origin 8.5.1 (OriginLab, Northampton, MA) software were used for data analysis. by hydrolysis of lidocaine. Therefore, we hypothesized that 21 activation of TRPV1 and/or TRPA1 may account for the Ratiometric [Ca ]i Measurements. Cells were stained by 3 mM Fura-2-AM and 0.01% pluronic for about 45 minutes. After injection pain caused by PPCM as well. We thus aimed to wash out to allow Fura-2-AM deesterification, coverslips were investigate whether activation of human (h)TRPV1 and/or mounted on an inverse microscope with a 20Â objective (Axio observer at ASPET Journals on September 24, 2021 hTRPA1 channels could account for injection pain caused by D1; Carl Zeiss, Jena, Germany). Cells were constantly superfused with intravenous PPCM application. We combined cellular meth- extracellular solution at room temperature containing 145 mM NaCl, ods including whole-cell patch clamp and calcium imaging. 5 mM KCl, 1.25 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM Furthermore, we wanted to indirectly explore activation of HEPES using a software-controlled seven-channel, gravity-driven, epidermal C fibers by PPCM and PCM in human volunteers by common-outlet superfusion system. Fura-2 was excited using a quantifying injection pain and flare reaction measured by microscope light source and an LEP filter wheel (Ludl Electronic laser Doppler